Capillary Electrophoresis and Electroosmotic Flow
Capillary electrophoresis (CE) offers high-resolution separation of ionic analytes due to efficient heat dissipation. Typically, CE is performed in fused silica capillaries but may be performed in glass or plastic chips. Ionization of silanol groups results in generation of electroosmotic flow that is detrimental to high resolution separations and has to be suppressed by introducing static or dynamic coating. A number of coatings, both static and dynamic ones, were introduced into capillary electrophoresis (J. Horvath and V. Dolnik, Wall coatings in capillary electrophoresis. Electrophoresis, 22 (2001) 644-655). Low values of electroosmotic mobility can be measured by the Williams-Vigh method (B. A. Williams and G. Vigh, Determination of accurate electroosmotic mobility and analyte effective mobility values in the presence of charged interacting agents in capillary electrophoresis. Anal. Chem. 69 (1997) 4445-4451).
To remove analytes and other compounds adsorbed on the capillary wall, the capillary is typically washed with 0.1 M N NaOH. Nevertheless, a flush with a high-pH NaOH solution results in formation of a liquid sodium silicate layer on the inner fused silica capillary surface (K. Klepárnik Z. Malá, P. Ba{hacek over (c)}ek, Fast separation of DNA sequencing fragments in highly alkaline solutions of linear polyacrylamide using electrophoresis in bare silica capillaries. Electrophoresis. 2001, 22, 783-788). This liquid layer prevents any wall interactions. Therefore, the capillary has to flushed with an acidic solution to neutralize the liquid sodium silicate at the capillary surface. Typically, 0.1 M HCl is used to for this purpose.
Biopolymers are frequently denatured prior the CE separation. DNA is usually denatured with urea, formamide, methyl formamide, dimethyl formamide, ethyl formamide, and dimethyl sulfoxide. Proteins can be denatured with sodium dodecyl sulfate, lithium dodecyl sulfate, sodium lauroyl sarcosinate, sodium decyl sulfate, lauric acid, urea, thiourea, formamide, methyl formamide, dimethyl formamide, ethyl formamide, dimethyl sulfoxide.
Analytes separated by capillary electrophoresis can be detected online by various detection techniques. UV absorption and laser-induced fluorescence of fluorescently labeled analytes are the most frequently used detection techniques in capillary electrophoresis.
Dynamic Coatings to Suppress Electroosmotic Flow
Chiari (U.S. Pat. No. 6,410,668) disclosed copolymers of various derivatives of acrylamide and methacrylamide with various glycidyl group containing monomers to form a highly hydrophilic, dynamic coating that suppresses electroosmotic flow.
Madabhushi et al. (U.S. Pat. No. 5,567,292) disclosed copolymers uncharged water-soluble silica-adsorbing polymers for suppressing electroendoosmotic flow, selected from the group consisting of polylactams, such as polyvinylpyrrolidone; N,N-disubstituted polyacrylamides; and N-substituted polyacrylamides.
Borate Polymorphs and Borosilicate Glass
Borates form various structures depending on pH. Borates are incorporated in a structure with silica to form borosilicate glass (A. K. Varshneya, Fundamentals of Inorganic Glasses, Academic Press, Boston 1994).
Polyol Borate Complexes
(For the sake of simplification, we call polyol any compound that contains at least 2 hydroxyl groups in its molecule.)
M. van Duin et al. (M. van Duin, J. A. Peters, A. P. G. Kieboom and H. van Beldam, Studies on borate esters I. The pH dependence of the stability of esters of boric acid and borate in aqueous medium as studied by 11B NMR. Tetrahedron 40 (1984) 2901-29141) described esters generated between boric acid and glycol, glycolic acid, oxalic acid, and glyceric acid.
Taylor et al. (M. J. Taylor, J. A. Grigg anf I. H. Laban, Triol borates and aminoalcohol derivatives of boric acid: Their formation and hydrolysis. Polyhedron 15 (1996) 3261-3270) described formation of mono-chelates, bis-chelates and cage structure between borates and triols, specifically Tris, tris(hydroxymethyl)ethane, tris(hydroxymethyl)propane, and triethanolamine.
Sonoda et al. (A. Sonoda, N. Takagi, K. Ooi, T. Hirotsu, Complex formation between boric acid and triethanolamine in aqueous solutions. Bull. Chem. Soc. Jpn 71 (1998) 161-166) described formation of triethanolamine-borate complexes in aqueous solutions.
Yan et al. (J. Yan, G. Springsteen, S. Deeter, B. Wang, The relationship among pKa, pH, and binding constants in the interaction between boronic acids and diols- it is not as simple as it appears. Tetrahedron 60 (2004) 11205-11209) described the relationship between pKa of monosubstituted boronic acids and their substituents using a Hammett plot.
pH Gradients by Polyols and Borate Buffers
Troitsky et al. (G. V. Troitsky, V. P. Zav'yalov, I. F. Kirjukhin, V. M. Abramov and G. Ju. Agitsky, Isoelectric focusing of proteins using a pH gradient created by a concentration gradient of nonelectrolytes in solution. Biochim. Biophys. Acta 400 (1975) 24-31) developed a new method of generating pH gradient by generating a concentration gradient of polyols in the presence of boric acid.
Shukun et al. (S. A. Shukun, A. V. Gavryushkin, V. N. Brezgunov, and V. P, Zav'yalov, Protein separation in pH gradients using free-flow electrophoretic apparatus. II. The pH gradients formed by the concentration gradient of boric acid in solutions of borax and mannitol. Electrophoresis 6 (1985) 75-77) generated pH gradient 3.5 to 9.2 for free-flow electrophoresis by forming a concentration gradient of boric acid in solutions of borax and mannitol for protein separation by free-flow electrophoresis.
Murel (U.S. Pat. No. 4,925,545) disclosed a method for forming pH gradients for use in electrophoresis and isoelectric focusing. The method generates a concentration gradient of polyols that interact with boric acid, borax, and/or other borate constituents and form acidic complexes with the anchored polyhydroxyl groups, generating pH gradient.
Borate Buffer for Electrophoresis of Carbohydrates and Polyols
Honda et al. (S. Honda, S. Suzuki, A. Nose, K. Yamamoto and K. Kakehi, Capillary zone electrophoresis of reducing mono- and oligo-saccharides as the borate complexes of their 3-methyl-1-phenyl-2-pyrazolin-5-one derivatives. Carbohydrate Res. 215 (1991) 193-198) separated derivatized aldopentoses and aldohexoses by CE in 100 mM borate buffer, pH 9.5.
Hoffstetter et al. (S. Hoffstetter-Kuhn, A. Paulus, E. Gassmann, and H. M. Widmer, Influence of borate complexation on the electrophoretic behavior of carbohydrates in capillary electrophoresis. Anal. Chem. 63 (1991) 1541-1547) disclosed the use of borate complexation for improving separation of carbohydrates in CE.
Stefansson and Novotny (M. Stefansson, M. Novotny: Separation of Complex Oligosaccharide Mixtures by Capillary Electrophoresis in the Open-Tubular Format Anal. Chem., 1994, 66, 1134-1140) described the use of borate buffer for separation of polysaccharides.
Landers et al. (J. P. Landers, R. P. Oda, M. D. Schuchard, Separation of boron-complexed diol compounds using high-performance capillary electrophoresis. Anal. Chem., 64 (1992) 2846-2851) separated diols with borate as counter ion.
Plocek and Chmelik (J. Plocek. and J. Chmelik, Separation of disaccharides as their borate complexes by capillary electrophoresis with indirect detection in visible range. Electrophoresis 18 (1997) 1148-1152) separated disaccharides by CE with indirect detection, when the concentration sensitivity for sucrose was 2 mM in 200 mM borate.
Quirino and Terabe J. P. Quirino and S. Terabe, Sweeping of neutral analytes via complexation with borate in capillary zone electrophoresis Chromatographia 53 (2001) 285-289) concentrated monosaccharides, catechols, and nucleosides by sweeping these analytes with borate.
Tris Borate Buffer for Electrophoresis of Glycoproteins
Weitzman et al. (S. Weitzman, V. Scott and K. Keegstra, Analysis of glycopeptides as borate complexes by polyacrylamide gel electrophoresis. Anal. Biochem. 97 (1979) 438-449) developed a new method of polyacrylamide gel electrophoresis in a Tris-borate buffer to analyze glycopeptides. The resolution of glycopeptides depended on the inclusion of borate ions in the sample, the gel, and the electrophoresis buffer. The borate ions reacted with neutral sugars, converting them into charged complexes which migrated during electrophoresis.
Keo et al. (U.S. Pat. No. 5,599,433) disclosed a buffer for capillary electrophoresis of glycosylated proteins comprising sodium borate and 3-cyclohexylamino-1-propanesulfonic acid.
Tris Borate Buffer for Electrophoresis of Nucleic Acid
Peacock and Dingman (A. C. Peacock, C. W. Dingman, Resolution of Multiple Ribonucleic Acid Species by Polyacrylamide Gel Electrophoresis. Biochemistry 6 (1967) 1818-1827) described electrophoresis of ribonucleic acid in polyacrylamide gels and resolution of multiple RNA species by polyacrylamide gel electrophoresis with Tris borate EDTA and boric acid buffers for electrophoresis in samples without glycerol.
Fuller (U.S. Pat. Nos. 5,314,595, 5,830,642, and 5,849,166) disclosed a method and a separation medium for capillary electrophoresis of DNA fragments containing glycerol, dithiothreitol (DTT) and trehalose or other sugars gel in the presence of a buffer lacking boric acid.
Carninci et al. (P. Carninci, S. Gustincich, S. Bottega, C. Patrosso; G. Del Sal, G. Manfioletti, C. Schneider, A simple discontinuous buffer system for increased resolution and speed in gel electrophoretic analysis of DNA sequence. Nucleic Acid Res. 18 (1990) 204-208) described a standard sequencing gel system using Tris/Borate/EDTA buffer (TBE). They also described a discontinuous buffer system using Tris-sulphate and Tris-borate. The Tris-sulphate was used as a running gel buffer, and Tris-borate as a tank buffer.
Strege and Lagu (M. Strege and A. Lagu, Separation of DNA restriction fragments by capillary electrophoresis using coated fused silica capillaries. Anal. Chem. 63 (1991) 1233-1236.) separated DNA restriction fragments in coated capillary in 0.5% methylcellulose, 50 mM Tris-borate, pH 8.0.
Brumley and Smith (R. L. Brumley and L. M. Smith, Rapid DNA sequencing by horizontal ultrathin gel electrophoresis. Nucl. Acids Res. 19 (1991) 4121-4126) describe the use of a borate buffer for a sequencing gel.
Bashkin et al. (J. Bashkin, M. Marsh, D. Barker, R. Johnston, DNA sequencing by capillary electrophoresis with a hydroxyethylcellulose sieving buffer. Appl. Theor. Electrophor. 6 (1996) 23-28) used TBE buffer containing 89 mM Tris, 89 mM boric acid, 1 mM EDTA with HEC to separate DNA sequencing fragments in a coated capillary.
Schwinefus and Bloomfield (J. J. Schwinefus, V. A. Bloomfield, The greater negative charge density of DNA in Tris-borate buffers does not enhance DNA condensation by multivalent cations. Biopolymers 54 (2000) 572-577) described the effect of Tris borate on the binding of multivalent cations to DNA.
Stellwagen et al. (N. C. Stellwagen, C. Gelfi and P. G. Righetti, DNA and buffers: the hidden danger of complex formation. Biopolymers 54 (2000) 137-142) described formation of borate-DNA complexes in polyacrylamide gels. They did not observe them in agarose gel due to the competition of agarose fibers for the borate residues.
Righetti et al. (P. G. Righetti, C. Gelfi and M. R. d'Acunto, Recent progress in DNA analysis by capillary electrophoresis. Electrophoresis 23 (2002) 1361-1374) studied interactions of DNA and small ions particularly borates. They were not able to answer the question, whether or not borate ions would bind to DNA.
Brody and Kern (J. R. Brody and S. E. Kern, Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis, BioTechniques 36 (2004) 214-216) concluded sodium borate is a better buffer than Tris borate for slab gel electrophoresis of DNA.
Buchmueller and Weeks (K. L. Buchmueller and K. M. Weeks, Tris-borate is a poor counterion for RNA: a cautionary tale for RNA folding studies. Nucleic Acid Res. 32 (2004) e 184) described difficulties related to the use of Tris borate buffer for electrophoresis of RNA.
Karger et al. (U.S. patent application 20040222095) disclosed the use of dimethyl sulfoxide for denaturation of DNA in capillary electrophoresis.
Singhal et al. (H. Singhal, Y. R. Ren and S. E. Kern, Improved DNA Electrophoresis in conditions favoring polyborates and Lewis acid complexation. PLOS one 5 (2010) e11318) proposed formation of polyborates and their complex formation with DNA.
Tris Borate Buffer for SDS Electrophoresis
Poduslo (J. F. Poduslo, Glycoprotein molecular-weight estimation using sodium dodecyl sulfate-pore gradient electrophoresis: Comparison of Tris-glycine and Tris-borate-EDTA buffer systems. Anal. Biochem. 114 (1981) 131-139) compared the accuracy of molecular-weight estimates for glycoproteins by SDS pore gradient electrophoresis in a Tris-glycine buffer system and Tris-borate-EDTA buffer and found the latter more accurate.
Wu and Regnier (D. Wu and F. E. Regnier, Sodium dodecyl sulfate-capillary gel electrophoresis of proteins using non-cross-linked polyacrylamide, J. Chromatogr. A, 608 (1992) 349-356) separated proteins by SDS CSE in bare capillaries with linear polyacrylamide as a sieving polymer. They prepared their protein samples in 100 mM Tris 250 mM borate with SDS, but did not disclose the composition of the sieving medium.
Zhang et al. (Y. Zhang, H. K. Lee and Sam F. Y. Li, Separation of myoglobin molecular mass markers using non-gel sieving capillary electrophoresis. J. Chromatogr. A 744 (1996) 249-257) described SDS capillary electrophoresis in bare capillaries using 12% dextran, 0.4 M Tris borate, 0.1% SDS, and 10% glycerol.
Harvey et al. (M. D. Harvey, D. Bandilla and P. R. Banks, Subnanomolar detection limit for sodium dodecyl sulfate—capillary gel electrophoresis using a fluorogenic non covalent dye. Electrophoresis 19 (1998) 2169-2174) performed SDS capillary electrophoresis in separation medium containing 8% linear polyacrylamide, 0.1 M Tris, 0.25 M borate, pH 8, and 0.05% SDS.
Bean and Lockhart (S. R. Bean and G. L. Lockhart, Sodium dodecyl sulfate capillary electrophoresis of wheat proteins. 1. Uncoated capillaries J. Agric. Food Chem. 47 (1999) 4246-4255) found optimum composition for SDS capillary electrophoresis in bare capillaries 10% dextran, 400 mM or 600 mM Tris borate buffer (pH=8.5), 0.1% SDS, and 10% ethylene glycol.
Liu et al. (U.S. patent applications 20040050702 and 20090314638) disclosed a sieving medium for SDS capillary electrophoresis of proteins comprising about 10% dextran, 600 mM Tris borate, 2 g/L SDS, 1 mM EDTA and 10% glycerol.
Other Electrophoretic Separations in Borate Buffer
Chen (F. T. A. Chen, Rapid protein analysis by capillary electrophoresis. J. Chromatogr. A 559 (1991) 445-453) separated serum proteins in 100 mM borate buffer pH 11.5 and 150 mM borate buffer, pH 10.5.
Alter and Kim (U.S. Pat. No. 5,753,094) disclosed a sample diluent for capillary electrophoresis based on a borate buffer.
Smith (U.S. Pat. No. 5,212,299) disclosed a composition for electrophoresis comprising glyceryl agarose and 20-400 mM borate.
Lee et al. (U.S. patent application 20090166200) disclosed a high-salt borate-buffered agarose gel for identification of a candidate vaccine.